While the use of embryonic mixed primary cerebellar cultures has proven valuable for dissecting structure: function relationships in Purkinje Neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published method is also very challenging. The focus of this study was to simplify the differentiation of mESCs into PNs. Using specific extrinsic factors, we successfully differentiated mESCs into PNs without the use of a postnatal feeder layer. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, mESC-derived PNs express the PN-specific markers Calbindin D28K, IP3R1, PLC4, and myosin IIB-B2. Using poly-l-ornithine, poly-l-lysine and Geltrex as the extra cellular matrix allowed mESC-derived PNs to be grown as monolayers, which is crucial for detailed live cell imaging. Staining of such monolayers with an antibody to the presynaptic marker Bassoon showed that granule neurons co-differentiate with PNs in mESC-derived cultures and form active synapses on PNs, akin to primary culture. As proof-of-principle, we introduced into mESC-derived PNs a custom, L7/PCP2 promoter-based plasmid to drive the expression of a miRNA against the motor protein myosin Va, which is required for the translocation of smooth ER into PN spines. Consistent with the effective knockdown of myosin Va, transfected mESC-derived PNs robustly phenocopied PNs isolated from dilute-lethal (myosin Va-null) mice with regard to the defect in spine ER inheritance. Together, the technologies described here should permit the development of a scalable, high-throughput method for dissecting specific molecular mechanisms in PNs, especially when a KO mouse is not available. Myosin X (MX) is a highly conserved, vertebrate-specific unconventional myosin whose tail domain contains a PIP3-specfic PH domain, a microtubule-binding MyTH4 domain, and an integrin-binding FERM domain. MX has been linked primarily to the formation and maintenance of filopodia (it is commonly referred to as the filopodial myosin), the transport of integrins in the plasma membrane, and the proper positioning of mitotic and meiotic spindles. Interestingly, neurons express both full length MX (FL-MX) and a headless version (Hdl-MX), and both appear to function in different aspects of radial glia migration (which give rise to most neurons and glia in the neocortex). MX has also been implicated in the transport of the netrin-1 receptor DCC to the tips of neurites, thereby regulating axonal path-finding. In our past efforts to define the function of another unconventional myosin (myosin Va) in Purkinje neurons (PN), the master neuron of the cerebellum, we developed novel tools to study this complex neuron. Interestingly, PNs express much higher levels of MX than other CNS neurons. Moreover, PNs are unique in undergoing filopodia-to-dendritic spine conversion without innervation, perhaps because they express high levels of this filopodial myosin. To begin to address the function of MX in PNs, we expressed GFP-tagged FL-MX in developing PNs. Time lapse imaging showed that MX localizes to the tips of dendritic filopodia, and then moves along these highly motile dendritic filopodia until it localizes to dendritic spines. To extend these observations, we have created a MX conditional knockout (cKO) mouse that targets both FL-MX and Hdl-MX. The whole-body MX KO shows partial embryonic lethality, and mice that survive exhibit a variety of defects including small size, fused digits and white belly spotting. Embryonic phenotypes include exencephaly and gross developmental defects. These data demonstrate that MX is critical for mouse embryogenesis, and that it probably plays a pivotal role in neural tube closure. To access the role of MX specifically within PNs, we are crossing our MX cKO mouse with L7-PCP cre mice, which express cre recombinase specifically in PNs. The mice obtained will be subjected to a variety of tests, from measuring animal coordination, to accessing PN structure and function in situ, in slices, and in culture. Together, these approaches should reveal the critical aspects of MX function in this complex neuron. Myosin Va, a class V processive motor, transports tubules of smooth endoplasmic reticulum (SER) into the dendritic spines of cerebellar Purkinje neurons (PNs). These SER tubules provide the source of calcium downstream of mGluR1 activation that drives synaptic plasticity at parallel fiber: PN synapses. Calcium release from SER tubules is mediated by the IP3 receptor (type 1 inositol 1,4,5-trisphosphate receptor or IP3R1), a resident SER protein. The focus of this study was to identify the receptor on the surface of the SER for myosin Va. A yeast two-hybrid screen and immunoprecipitation analyses identified two potential receptor components that interact with both myosin Va and IP3R1: phospholipase C beta 4 (PLC4) and receptor-for-activated-kinase 1 (RACK1). Initial work has shown that both PLC4 and RACK1 are enriched in dendritic spines, and live cell imaging has shown that RACK1, like myosin Va, localizes at the tip of SER tubules that are moving into spines. Furthermore, the dendritic spines of PNs from IP3R1-null mice are devoid of SER, arguing that IP3R1 is required for the myosin Va-dependent translocation of SER into spines. Finally, the enrichment of PLC4 and RACK1 in dendritic spines is disrupted in both myosin Va knockdown cells and IP3R1-null cells. Together, these data suggest that myosin Va may be recruited to the SER via the IP3 receptor, with PLC4 and/or RACK1 providing a bridge linking the myosin to the receptor.